Drosophila small nucleolar RNA (snoRNA), named jouvence whose loss of function reduces lifespan. The genomic region of jouvence rescues the longevity in mutant, while its overexpression in wild-type increases lifespan. Jouvence is required in enterocytes. In mutant, the epithelium of the gut presents more hyperplasia, while the overexpression of jouvence prevents it. Molecularly, the mutant lack pseudouridylation on 18S and 28S-rRNA, a function rescued by targeted expression of jouvence in the gut. A transcriptomic analysis performed from the gut reveals that several genes are either up- or down-regulated, while restoring the mRNA level of two genes (ninaD or CG6296) rescue the longevity. Since snoRNAs are structurally and functionally well conserved throughout evolution, we identified putative jouvence orthologue in mammals including humans, suggesting that its function in longevity could be conserved.Longevity is influenced by genetic and environmental factors, but the underlying mechanisms remain elusive. Here, we functionally characterise a  jouvence, which extends the lifespan of fruit flies through its function in the gut, and is conserved in humans.Small non-coding RNAs contribute to the regulation of aging. Here the authors identify a small nucleolar RNA, the snoRNA  Several genes and metabolic factors have been reported to modulate longevity16. Indeed, genes that increase longevity have been identified in different model organisms, such as yeast4, C. elegans17, Drosophila16 and mouse18. More particularly, in Drosophila, these include mathuselah5, insulin-signalling pathway genes15, Cu/Zn superdismutase9, sirtuin10, and genes affecting either the regulation of mitochondria as PGC-111 or the mitochondrial respiratory chain12. It is also well accepted that lifespan is environmentally modulated. In addition, longevity positively correlates with the ability to resist to stress. In such way, environmental factors like diet1, and stress14 also modulate an organism’s lifespan. More recently, some microRNAs (miRNAs) were identified as key regulators of genes involved among other processes as neurodegeneration, in aging21.The aging process is the result of complex biological mechanisms leading to accumulation of different types of damage at molecular, cellular, tissue and organ levels. This leads to the decrease or loss of certain physiological functions, and therefore, to an increase of vulnerability to diseases and death22. They are known to be present in the nucleolus, in which they are associated with a set of proteins to form small nucleolar ribonucleoprotein (snoRNPs)22. They are generally known to be processed from introns of pre-mRNAs. They are believed to have several functions, including the 2’-O-methylation and pseudouridylation of different classes of RNA, the nucleolytic process of the rRNA, telomeric DNA synthesis23, as well as they can target the spliceosomal snRNAs (whose are their main targets after rRNAs). Based on conserved secondary structure and functional RNA motifs, two major classes have been distinguished, the box C/D and box H/ACA. The box C/D, comprising notably the multiple “U” snoRNAs , generally performs the 2’-O-methylation of ribosomal RNA, while the box H/ACA directs the conversion of uridine to pseudouridine, and so particularly of rRNA23. However, recent studies have demonstrated that the H/ACA snoRNA might also pseudouridinylate other RNA substrates, such as mRNA and long-non-coding-RNAs (lncRNAs)24 or have a role in chromatin remodelling, indicating that they have several other functions25. Then, although few H/ACA box snoRNA have been involved in human diseases27, their role at the organismal level and more particularly in aging has not yet been documented.The snoRNAs are members of a group of non-coding RNAs. SnoRNAs are found in species from archeobacteria (in which they are named sRNAs) to mammalsjouvence (jou) and show that its deletion reduces lifespan. A transgene containing the genomic region of jou is able to rescue the lifespan, while its overexpression increases it. In situ hybridization (ISH) reveals that jou is expressed in the enterocytes, the main cell-types of the epithelium of the gut. At the cellular level, in jou-deleted flies, the number of enterocytes as well as the proliferative cells increase leading to an intestinal hyperplasia. Genetic targeted expression of jou in the enterocytes prevents the hyperplasia, which is sufficient to rescue and significantly increase longevity, and so, even when it is expressed only in adulthood . Finally, as snoRNAs are generally well conserved throughout evolution, both structurally and functionally, we identify putative jou mammalian orthologues, both in mouse and human, suggesting that it may have an implication in mammalian aging.Here, we characterize a small nucleolar RNA (snoRNA:Ψ28S-1153) that we name 30. In a study to characterize putative genes involved in locomotor activity and more particularly genes expressed in the central complex (CC), a premotor centre localised in the middle of the brain31, a P-element insertional mutagenesis was performed. We identified the enhancer-trap line P[GAL4]4C, and show that the blockage of the targeted ring neurons disrupts the power law distribution of walking time intervals32. Here, in a step further, we molecularly characterize the P[Gal4]4C line, as well as we study it in the context of aging and particularly in aging effects on sensory-motor performance. P[GAL4]4C is inserted on the second chromosome, at position 50B1  between CG13333 and CG13334 near the P[GAL4]4C insertion 35 localized just upstream to the first snoRNA:Ψ28S-1153. However, according to the canonical definition of snoRNA in which the H/ACA box is predicted to form a hairpin-hinge-hairpin-tail structure  (shortly named: Del)  Fig. , which cel) Fig. .Fig. 1Mo37. The deletion flies in a Berlin genetic background still live shorter than their co-isogenic Berlin flies . Longevity tests Fig.  performe34, we hypothesize that this snoRNA might be potentially the only true functional snoRNA in this locus. In order to determine in which tissues snoRNA:Ψ28S-1153 could be expressed and required to affect lifespan, we performed ISH on the whole flies using fluorescently-labelled probes , an enhancer trap in myosin 1A gene, encoding a gut specific brush border45. As described by Jiang et al.46, Myo1A-Gal4 is strongly expressed in all midgut ECs, while no expression was detected in ISCs, EBs, EEs, or visceral muscle. To label the EBs, we used the Su(H)GBE-Gal444, while the ISCs were marked by both esg-Gal4 and Dl-Gal4. Amongst these, only EC-specific expression , enteroblasts (EBs), entero-endocrine cells (EEs) and intestinal stems cells (ISCs)8M) transgenic line. Then, we targeted this unique snoRNA expression specifically to enterocytes in deletion background using Myo1A-Gal4 line  and verified by ISH that the snoRNA was indeed correctly expressed in the enterocytes   with a second Gal4 driver line, Mex-Gal447, which is also expressed in the epithelium of the gut though not exclusively in the ECs48 49 and generate a transgenic fly line (named pJFRC-MUH-jou1M) inserted at the attP2 site located at position 68A4 on the third chromosome. We then repeated the longevity experiments using the same P[Gal4] driver lines . Both of them lead to an increase of longevity  and confirm that the longevity effect is due to the snoRNA-Ψ28S-1153 in the epithelial cells of the gut, and not to the two other putative snoRNAs, we generated a p[UAST-snoRNA-Ψ28S-1153]    to allow expression of the UAS-joujouvence. We also quantify the level of the snoRNA on dissected gut . Similarly the Mex-Gene-Switch driving the snoRNA-jou  induced an increase of jouvence after RU486 induction (up to 5 folds)   : all three controls don’t show any difference in longevity comparing fed versus non-fed flies with RU486  (which means “youth” in English).Since in the deletion background, the targeted expression of the snoRNA:Ψ28S-1153 specifically in the enterocytes rescued the phenotype, we wondered if the overexpression of this snoRNA in a Wild-Type genetic background (CS) could be beneficial leading to an increase of lifespan or in contrast, be deleterious. We used the same driver lines to overexpress the snoRNA:Ψ28S-1153 up to 13-fold  and 28-fold  over that of endogenous expression Fig. . Both Ga51. Then, we wonder if the deletion of the snoRNA-jouvence affects the homoeostasis of the gut and leads to cellular lesions. To assess such hyperplasia, we count the number of enterocytes both in young (7 day-old) and old (40-day-old) flies. We observe that this number is increased in the jou-deleted flies compared to their respective WT controls . In addition, since the expression of jouvence (in the deletion background) in the gut is sufficient to rescue and even prevent the hyperplasia, we wonder if the overexpression of jouvence in a Wild-Type background could be sufficient to prevent such hyperplasia in WT flies. Figure 1M and Myo1A-Gal4>jou1M).In aged flies, the intestinal epithelium degenerates leading to intestinal hyperplasiaols Fig. , indicat52. Figure jou-deleted flies. Second, to assess the proliferation of the ISCs, we count the number of anti-PH3 positive cells51. Interestingly, it reveals  which labels the dividing as well as the endoreplicative cells. In Fig. jou-deleted flies (in accordance to the higher number of endoreplicative ECs), while in aged flies, the number of EdU cells is increased in WT but not statistically different in jou-deleted flies, which seems also to reflect quite faithfully the number of Dl positive cells  as well as the putative mis-differentiation of ISCs daughter cells (enteroblasts: EBs), using different labelling approaches. First, we use the Delta-Gal4 to drive the pUAS-GFP-nls to count the number of ISCsals Fig.  a quite lls Fig. . AltogetDrosophila, several tissues, as gut, fat body, salivary glands, Malpighian tubules, trachea, muscle and epidermis contain polyploïd cells due to endoreplication cycle55. The ISH has revealed that the snoRNA-jouvence is specifically expressed in two endoreplicative cell types, the enterocytes and ovarian nurse-cells, but not in other endoreplicative tissues. Thus, we wonder if the endoreplication is affected in jou-deleted flies. We then assess, by two independent and commonly used approaches, the endoreplication in the gut. First, we quantify the amount of DAPI-staining fluorescence of the nucleus, which reflect the quantity of DNA56. Figure jou-deleted flies compared to WT-Controls, indicating less endoreplication. Interestingly, the quantity of DNA (endoreplication) is rescued in flies bearing the genomic-rescued transgene (Del+rescue-1), as well as in deletion flies with a targeted expression of jouvence specifically in the gut . In a step further, we also quantify the endoreplication in flies overexpressing the snoRNA-jouvence in WT genetic background, and more particularly when expressed only in adulthood using the Gene-Switch system. After feeding RU486 to induce the jouvence expression, the endoreplication is increased compared to the non-fed RU486 control flies (Mex-GS>jou1M), indicating that jouvence is involved in the control of the polyploidy in ECs. This last result also demonstrates that jouvence, among the three snoRNAs of the locus, is sufficient by itself to modulate the endoreplication. Second, to reinforce such measurement, we also quantify, by RT-qPCR, the Cyclin-E, one of the main genes known to be involved in the endoreplicative cycle57  or in flies with specific targeted expression of jouvence in the gut .Though we have shown by RT-PCR that 34, the snoRNA-jouvence (snoRNA:Ψ28S-1153) is predicted to catalyse the modification of a 28S-rRNA base, at position 1153. Based on the tissue-specific expression of jou, and to grasp insights into the molecular mechanism of action of jou, we check if the rRNA, extracted from the gut, are indeed pseudouridylated. Figure jou-deleted flies, indicating that jou is required for this site specific pseudouridylation. Moreover, the re-expression of jouvence in jou-deleted flies carrying the genomic-rescued transgene (Del+rescue-1) as well as in flies with specific targeted expression of jouvence in the gut  demonstrates that the pseudouridylation mechanism is rescued. Additionally, we also show that jouvence pseudouridylates the 18S-rRNA at position 1425 in WT control flies  and gut specific targeted expression .According to the reported bioinformatic study in which the snoRNA:Ψ28S-1153 has been primarily annotatedies Fig. , which ijou, and to better understand jou function in longevity we performed a transcriptomic analysis (RNA-Sequencing) of the gut tissue comparing Wild-Type to jou-deleted flies. Based on a standard stringency of 2-fold changes, RNA-Seq reveals that 314 genes were upregulated, while 319 genes were downregulated , Gba1a (Glucocerebrosidase 1a), LysB (Lysozyme B), and ninaD  are strongly upregulated in deletion line, whereas CG6296 (lipase and phosphatidylcholine 1-acylhydrolase predicted activities), and Cyp4p2 (Cytochrome P450-4p2) are strongly downregulated, confirming the results of the RNA-Seq. The regulation of these genes by jou was confirmed by the analysis of the rescued genotypes. For GstE5 gene, the level of RNA is rescued in genomic-rescued line (Del+rescue-1) as well as in Myo-Gal4 expressing jou in deletion background  specifically in enterocytes. For Gba1a and LysB, the rescue is quite similar to GstE5. However, for the ninaD, the regulation seems to be more complex, since the level of RNA is inverted (importantly increased) in genomic-rescued line  and not rescued in Del,Myo1A>UAS-jou8M line, suggesting that for this gene the fine regulation of the RNA level is more complex and might putatively involve other factors. For the two selected downregulated genes (CG6296 and Cyp4p2), similar to ninaD, the regulation seems also to be complex since none of them are rescued by the genomic-rescue line  neither by the Myo1A-Gal4 , although it is partially rescued  in Del,rescue-1 and Del,Myo1A>UAS-jou8M for CG6296. These results suggest that the longevity function of the snoRNA-jou relies on its ability to regulate either the mRNA level or the ribosomal RNA stability, or even perhaps the gene expression in the enterocytes. However, such precise mechanisms remain to be further studied.To confirm some of the results obtained by the RNA-Seq analysis, we selected some of the most up- or downregulated genes to perform RT-qPCR on gut tissue. Figure jou-deleted flies, which leads to a shorten lifespan. Inversely, we wonder which genes are modified in flies overexpressing jou in a WT genetic background, whose yields to an increase of lifespan. Thus, we performed a second RNA-Seq on the dissected gut of flies expressing conditionally jouvence only in adulthood using the Gene-Switch system (Mex-GS>jou8M), comparing RU486-fed flies (induced expression) with no-RU486 fed flies (control-non-induced). Surprisingly, it reveals that only 9 genes are deregulated in jouvence overexpressing flies, based on a standard stringency of 2-fold changes , to direct a RNAi, in the aim to decrease and restore its mRNA level. We show that the targeted expression of a ninaD-RNAi specifically in the gut, and more precisely only in adulthood , is sufficient to importantly decrease and restore the mRNA level 59. Then, we wonder whether jouvence could affect resistance to stress. To elucidate jou’s role in stress response, we tested the resistance of transgenic lines (Del+rescue-1 and rescue-1) to desiccation and starvation. During the desiccation test, young flies were placed at 37 °C without water, and their survival time was quantified  flies , respectively  revealed jou homologues in 12 Drosophila species, two orthologues in mice and one in human , which are highly homologous (more than 90%)  were identified just upstream to the snoRNA jouvence in 11 over the 12 other Drosophila species, except in Drosophila grimshawi (the most divergent from D. melanogaster), in which only the sno-2 is present. However, these two putative snoRNAs have not been identified upstream to the jou homologue in mammals, suggesting that these two snoRNAs are likely not directly functionally linked to jouvence.The snoRNAs are known to be well conserved, both structurally and functionally, throughout evolution from archaebacteria to humansman Fig. , suggestjouvence in mammals, we assessed expression by RT-PCR in various human and mouse tissues . Finally, as snoRNAs are known to be generally located in large introns, we wonder if the genes in which the snoRNA is located or in the vicinity are expressed. In human, the snoRNA jouvence is located in a long intron of the TEAD1 (TEA domain family member 1) gene , whose deletion decreases lifespan, while inversely, its overexpression increases it. Several independent genetic experiments support this conclusion. First, three independent genomic-rescued transgenic lines  rescue the longevity in deletion line. Second, since ISH revealed that jou was expressed in the enterocytes of the epithelium of the gut, targeted specific expression of solely jou snoRNA in these specific cells, using two independent gut specific P[Gal4] driver lines  and both combined to two independent UAS-jouvence transgenic constructs (UAS-jou8M and pJFRC-MUH-jou1M) were sufficient to rescue the longevity in the deletion, and even more precisely, only in adulthood (through the use of the Gene-Switch system) (Mex-GS). Moreover, Northern Blots analysis reveal that jou is detected in the gut at the expected size (148 nucleotides), validating its expression and processing. Finally, to extend our general characterization of the snoRNA-jouvence, RT-qPCR have been performed at different developmental stages revealing that jouvence is faintly expressed in all stages, except with a slight stronger expression in the white pupae  that we have named jouvence specifically in the enterocytes  was sufficient to rescue the mRNA level of several of them, demonstrating that jou is the snoRNA responsible for these deregulation. Moreover, among these genes, several are involved in stress resistance. Thus, while deletion line was more sensitive to stress, the rescue of jou in deletion increases the resistance to these stress, demonstrating that the presence of jou in the gut is sufficient to both increase lifespan and protect against various stress.Transcriptomic analysis performed on deletion line has revealed that several (few hundred) genes are deregulated. Several of those genes have been validated by RT-qPCR, while the genetic rescue of 35 has revealed two other putative snoRNAs, localized just upstream to the snoRNA:Ψ28S-1153 (jouvence). Since the DNA-fragment of 1727 bp used to build the genomic-rescue  also contains these two putative snoRNAs, thus a putative effect of these two snoRNAs could not be excluded. To verify and rule-out that the longevity effect was not due to these two putative snoRNAs, we have targeted the expression of the each of them  in the enterocytes. The expression of the each of these snoRNA individually (in the deletion background) did not rescued or lead to an increase of longevity compared to their co-isogenic control lines, confirming that the longevity effect is due to the snoRNA-jouvence.However, while our study was ongoing, a general transcriptomic analysisjou acts in the fly gut epithelium to promote longevity. In Drosophila, a relationship between lifespan and the gut have been previously reported60. In old Wild-Type flies, ISCs over-proliferation causes mis-differentiation of ISC daughter cells, resulting in intestinal hyperplasia: a phenomenon called “hyperproliferative diseases”. Interestingly, preserving hyper-proliferation in the gut epithelium extends lifespan51. We have characterized both in control WT and deletion line, and so both in young and old flies, the number of different cell types in the gut. As reported, the number of ECs are increased in old WT flies, confirming a hyperplasia. Whereas, this increase is higher in jou-deleted flies, indicating more hyperplasia. However, remarkably, the targeted expression of jouvence in the gut, by two independent P[Gal4] driver lines , as well as the overexpression  of jou prevents this hyperplasia. To point-out more precisely the origin of this hyperplasia, three different and independent quantitative analysis based either on a genetic marker , or immuno-histological staining  have been performed and the labelled cells counted in the overall gut , but we did not notice any significant difference between the Control-WT and the deletion flies .Our findings indicate that C. elegans, a link between the nucleolus size and lifespan has been established61, a mechanism that has been related to the nucleolar protein fibrillarin. The fibrillarin protein is associated to the RiboNucleoProtein (RNP) of the C/D box snoRNA, whereas the snoRNA jouvence belongs to the H/ACA box, which does not involve the fibrillarin. Nevertheless, in some ways, our study extends these findings61 by specifically identifying a specific cell population (enterocytes) and a specific snoRNA (jouvence), reinforcing this previous finding that the nucleolus is involved in the determination of longevity.Recently, in 14. Interestingly, loss of jou function decreases resistance to desiccation, while inversely, the rescue or overexpression of jou increased resistance to this stress test. These protective effects are likely due to the increase of the Hsp78Bb gene expression level revealed by the RNA-Seq performed on overexpressing jouvence flies, a hypothesis that remain to be tested. However, in response to starvation, the jou-dependent resistance was found to be altered depending on the age of the fly . Such a difference and even opposite effect in resistance to various stress tests has previously been reported28, suggesting that the effect on the stress resistance depends rather of the stress test itself rather than being a universal rule of the longevity genes. Indeed, each stress test relies on specific physiological and/or metabolic parameters that might be differently solicited for each stress. Dessiccation is mainly an indicator of the water physiology/homeostasis while the starvation is principally an indicator of the energy metabolism (lipids and/or glucose). In addition, since starvation effect of jou deletion changes during the adult lifespan (increased in young but reduced in old flies compared to controls), this could suggest that energy metabolic components are modified or altered during the course of the adult life.Many longevity genes have been shown to modify the resistance to stress testsjouvence, the RNA-Seq and RT-qPCR analyses revealed that several genes are either up- or down-regulated in the deletion line. Interestingly, the first striking outcome of the overview of the 633 differentially expressed genes , are not strikingly affected, except the dILP3  deregulated. The second striking observation revealed by the RNA-Seq concerns the reported link between the Myc/ribosomes biogenesis and lifespan, either in C. elegans62, Drosophila63, and mouse64. Indeed, it has been reported that several snoRNAs are a class of Myc targets65. However, though this study conducted on S2 cells has identifyed about 500 Myc target genes, whose function mainly resides in the control of ribosome biogenesis, the snoRNA jouvence itself has not been identified in this group of genes regulated by Myc. This result is corroborated by the fact that we did not found any Myc consensus E-box sequence (CACGTG)65 in the vicinity of the jouvence gene (the nearest one being located at 3112 bp in 5’), as well as the Myc gene and the ribosomes biogenesis pathways are not affected in deletion line 66 instead of acting directly on ribosome synthesis.To initiate deciphering the molecular mechanism of the snoRNA-es over 94 deregulGstE5, Gba1a, and LysB), the level of RNA was rescued by the re-expression of jouvence in the enterocytes, demonstrating that jou is clearly the snoRNA responsible of this deregulation. However, for some other genes  the deregulation seems to be more complex, either requiring a very precise level of snoRNA-jou, or potentially involving the two other putative snoRNAs. To identify and confirm a link between the snoRNA-jouvence, the deregulated genes, and the longevity phenotype, we restore in the deletion genetic background, for two of them (ninaD and CG6296), the level of their transcripts. Remarkably, decreasing the level of ninaD  by the expression of a RNAi specifically in the gut, or inversely increasing the level of CG6296 (a downregulated gene) by the expression of its cDNA, increase lifespan, demonstrating that these two genes, even taken individually, are crucial for the determination of lifespan  metabolism and in maintaining plasma cholesterol levels. CG6296 (a non yet well characterized gene) presents protein domains/motifs similar to triacylglycerol lipase family, with predicted lipase activity and phosphatidylcholine 1-acylhydrolase activity, suggesting a role in lipids metabolism (FlyBase). Taken together, these results indicate that the snoRNA jouvence is involved in the fine tuning of the level of many mRNAs. We have demonstrated that jouvence pseudouridylates the 28S-rRNA and 18S-rRNA as predicted by its primary sequence34, a function restored by the various transgenic lines. Therefore, the snoRNA-jouvence might likely act either in the maturation and/or stabililisation of the rRNA, although an involvement in the regulation of the transcription or even potentially in the chromatin structure, as already reported for some other snoRNAs25 could not at this stage of our study, be excluded. Interestingly, as reported for the methylation of ribosomal RNA by NSUN5, in which the modification of a single rRNA nucleotide yields to functionally different ribosomes66, here, the pseudouridylation of a specific base on rRNA (Ψ at position 1153 on 28S-rRNA) potentially generates a specialized ribosome. Further experiments, as for example, the determination of the 3D structural conformation of the ribosomes (pseudouridylated or not) will be required to answer this precise question. Finally, although it is commonly considered that the snoRNAs might mainly be an ubiquitous and housekeeping machinery in all cells, obviously, as for the methylation of ribosomal RNA by NSUN566, our results rather argues for a specific snoRNA machinery in certain tissues, as for instance here, in the epithelium of the gut.Moreover, some of these genes have been validated by RT-qPCR. In addition, for some of them  yields a pleiotropic genetic disease, the congenital dyskeratose, in which patients have shorter telomeres27. It has also been reported that the snoRNA HBII-52, a human C/D box type of snoRNA, regulates the alternative splicing of the serotonine receptor 2C67. Therefore, we speculate that the mammalian jou orthologue could play important functions in mouse and human respectively, but it remains to be investigated. Nevertheless, the conservation of sequence and the expression of jou raise the exciting hypothesis that similar functions may also be conserved in vertebrates including humans.Based on its primary sequence Fig. , jou canjou represents a good candidate to understand the relationship between the gut, the aging, and the longevity. Interestingly, as already reported, the intestinal cells are very similar in Drosophila and mammals, both at cellular and molecular levels68, while snoRNAs are well conserved throughout evolution22. Although jouvence was not yet annotated in the mammalian genome including human, we have shown by two independent and complementary approaches (RT-PCR and Northern Blot) that jou is present in mammals and is expressed in the gut (among other tissues), strongly suggesting that it might be functional. Therefore, we could hypothesize that manipulating the mammalian gut epithelium may also protect against deleterious effects of aging and even increase longevity (including in humans). Thus, jou snoRNA could represent a promising therapeutic candidate to improve healthy aging.Thus, undoubtedly, Drosophila melanogaster lines were maintained at 24 °C, on our standard dried brewer yeast and cornmeal medium (without sugar or molasses), in a 12 h light/12 h dark cycle. Canton Special (CS) flies were used as Wild-Type control. The deletion (F4) (short named: Del) and the genomic rescue transgenic flies  were outcrossed a minimum of 6 times with CS (Cantonization) to thoroughly homogenize the genetic background. To follow the deletion (F4) during the Cantonisation (and similar for the Berlinisation), PCR has been performed at each generation to re-identify the fly (chromosome) bearing the mutation (genotyping). The Gal4 lines used , as well as the generated transgenic lines  were also backcrossed 6 times with CS (Cantonization). The two Gal4 lines  were then crossed to each of the snoRNA transgenic construct lines to determine their longevity, while in parallel, they were crossed to deletion (F4) to generate the co-isogenic control flies . The lines Myo1A-Gal4 (located on chromosome 2), esg-Gal4 (chromosome 2), were kindly provided by B.A. Edgar, Heidelberg, Germany. The lines Su(H)GBE-Gal4 (chromosome X), and Dl-Gal4 (chromosome 3) were a courtesy of X. Zeng, NCI/Frederick, USA. Mex-Gal4 (chromosome 2) was kindly provided by G.H. Thomas, Pennsylvania State Univ., PA, USA. The UAS-ninaD-RNAi line (31783R) has been provided from NIG (Japan). This line has also been backcrossed 6 times with CS (Cantonization), and thereafter introduced in the deletion background by standard genetic crosses.Two primers located at positions 66851F and 68140R  and the reverse primer (snoRNAgenomic-R) (see XbaI cloning site at the 5’-ends. The amplified fragment was then inserted via the Xba1 restriction site into the pCaSper 4 (which contains no enhancer/promoter sequence). This way, snoRNA expression is dependent on its own genomic regulatory sequences included in the inserted DNA fragment. The transgenic flies were generated by standard transgenic technique , and three lines have been obtained . Rescue-1 and rescue-2 are inserted on the third chromosome, while the rescue-3 is inserted on the second chromosome.The 47 has been inserted through the EcoR1 and DraII sites into the pattB-Sf-HS-GALGS (Sfi1 orientated) . The transgenic flies have been generated by BestGenes (USA), in which the plasmid-construct has been inserted in VK02 site located at position 28E7 on the second chromosome. The Mex-GS transgenic line has been re-introduce in the deletion background by standard genetic cross and recombination.To generate the Mex-Gene-Switch line, we use the forward primer: Mex1-F + a Sfi1 cloning site at the 5’-ends: 5’-ATAGGCCGGACGGGCCAACGCGAATTCAGACTGAGC-3’, and the reverse primer: Mex1-R + a Sfi1 cloning site: 5’-TGTGGCCCCAGTGGCC CGTTGCACATGGTGATGACT-3’, to amplify by PCR using the genomic DNA, a fragment of 2264 bp. Then, this DNA fragment containing the regulatory/promoter sequence of the Mex gene, as formerly reportedjou of 148 bp was cloned into the TOPO-TA cloning vector (Invitrogen) using the same primers used for RT-PCR (see Suppl. Information for snoRNA-Fb and snoRNA-Rb). This construct was also used to synthesize probes for ISH. First, the snoRNA fragment of 148 bp was subcloned into the p[UAST] vector. Second, similarly, the snoRNA fragment of 148 bp was subcloned into the pJFRC-MUH vector49  containing the attB insertion sites allowing a directed insertion. Similarly, for the cloning of the DNA fragment containing the sno-2 or the sno-3, appropriated forward and reverses primers . We then use the forward primer: CG6296F + a BglII cloning site at the 5’-ends: 5’-TTCACTTCAACGATGAGATTGAGATCT-3’, and the reverse primer: CG6296R + a XbaI cloning site: 5’-TCTAGAGGACTTGCAATATTATTTGA-3’, to amplify by PCR a fragment of 2167 bp. Then, this cDNA fragment has been inserted through the BglII and XbaI sites into the pJFRC-MUH vector. The transgenic flies have been generated by BestGenes (USA), in which the plasmid-construct has been inserted in VK27 site located at position 89E11 on the third chromosome. Thereafter, the pJFRC-MUH-cDNA-CG6296 transgenic line has been backcrossed 6 times with CS (Cantonization), and then introduced in the deletion background by standard genetic crosses.Drosophila, total RNA from whole female flies was extracted by standard procedures using Trizol (Invitrogen). This was treated with RQ1 RNase free DNase (Promega) to remove any DNA contamination, and verified by PCR. Following this, RT was performed with MMLV Reverse Transcriptase (Invitrogen) using a random primer hexamer (Promega), followed by a 20-min RNase H (Invitrogen) at 37 °C. The PCR was performed using forward and reverse primers , with Taq DNA Polymerase (Invitrogen), at 51.4 °C. Similar procedures were used to amplify the 300 bp control RP49 fragment using the forward and reverse primers respectively. Mouse and human total RNA were commercially purchased at Amsbio: Mouse Brain, whole Total RNA: MR-201, Mouse Ovary Total RNA: MR-406, Mouse Kidney Total RNA: MR-901, Human Brain Total RNA: HR-201, Human Ovary Total RNA: HR-406, Human Kidney Total RNA: HR-901. The human gut RNA was purchased at Biochain: Human adult Normal small Intestine Total RNA: T1234226. All of these total RNA were re-treated by the DNAse to completely remove the DNA, and re-verified by PCR before performing the RT-PCR. Thereafter, RT-PCR have been performed in similar conditions as for Drosophila. For the sequence primers, see For Drosophila, total RNA were extracted from 30 dissected gut as described above for the detection of jou expression, followed by RT-PCR. For the Drosophila Cyclin-E, see jouvence orthologue detection (see above). For the TEAD1 (human), CNNT and EPC1 (mouse), see For jou. The TaqMan Universal Master Mix II, no UNG was used with a specific Taqman probe designed for the snoRNA jouvence and a commercial Taqman probe for RP49 . The amplification was done an a BioRad apparatus (Biorad CFX96) with a program set at: activation 95 °C, 10 min, PCR 40 cycles: 95 °C, 15s, 60 °C, 1 min. Then, the ratio of the snoRNA (dilution 1/5) was reported to the RP49 (dilution 1/100 - since the RP49 is more expressed) and then calculated and normalised to CS = 1. Taqman RT-qPCR was performed in duplicate on each of 3 or 5 independent biological replicates, depending of the genotype. All results are presented as means ± S.E.M. of the biological replicates.For RNA extraction of whole flies or dissected gut, similar procedures described above for RT-PCR were used. The samples were diluted in a series of 1/5, 1/10, 1/100 to establish the standard curve, then 1/100 was used for the internal control (RP49) and 1/5 for TM). On d3, the sections were washed four times at 45 °C: 3/0 - 3/1 - 1/1 - 1/3 with hybridization buffer/PBS, 15 min each wash. Sections were then washed four times with PBS 1X 0.1% tween, 5 min at RT, and PBS 1X 0.1% tween + 10% goat serum, 1 h, followed by the incubation with the primary antibody, anti-Digoxigenin-mouse-HRP conjugate at 1/100  for 1h, RT. The immuno-reaction was amplified with the tyramide amplification kit  for 8 min in the dark; the sections were washed as usual and counter-stained with DAPI (Roche) for 5 min. The preparation was mounted with Mowiol. For double labelling, following tyramide signal amplification, the sections were incubated with goat serum 2% in PBS 1X 0.1% Tween-20, for 2 h at RT, and incubated with anti-GFP at 1/500 , overnight. On d4, the slides were washed, and incubated with the secondary antibody, anti-mouse FITC at 1/500 , washed as usual, and mounted with Mowiol. Same protocol has been used for the ISH on dissected gut, except that we use a FITC-labelled tyramide instead of a Cy3-labelled tyramide.For whole flies ISH, we used 5 day-old females. On day-1 (d1), whole flies were fixed with 4% paraformaldehyde (PFA) for 6 h at 4 °C, and then incubated overnight in 25% sucrose solution at 4 °C. On d2, individual flies were embedded in 20% carboxymethylcellulose, frozen in liquid nitrogen, and sectioned using a Cryostat (−20 °C) at 30 μm thickness. The sections were rehydrated for 15 min in PBS-1X + 0.1% Tween-20, and post-fixed with 4% PFA for 15 min. They were washed twice with PBS-1X + 0.1% Tween-20, and treated with Proteinase-K (10 µg/ml) for 5 min. Proteinase-K was inactivated using glycine (2 mg/ml) and the sections were washed and post-fixed again. For ISH, the fixed sections were washed with hybridization buffer  diluted with PBS (1/1) followed by pre-hybridization for 1h at 65 °C using the same hybridization buffer. Overnight hybridization with the snoRNA anti-sense probe (or sense as negative control) was performed at 45 °C. The sense and anti-sense probes were synthesized using the DIG RNA labelling kit (SP6 or T7)  and then pre-hybridized for 4 h in 50% formamide, 5X SSC, 5X Denhardt, 50 mM sodium phosphate pH 7.5, and 0.1% SDS (w/vol), and 100 μg ml−1 of denatured DNA from salmon sperm, before an overnight hybridization under the same conditions. Pre-hybridization and hybridization were carried out at 42 °C. The day after, the membrane were washed as follows: a spontaneous wash with 4X SSC during 5 min at RT, followed by three successive washes of 4X SSC, 0.1% SDS, 1X with 1X SSC, 0.1% SDS, and finally 1X with 0.5X SSC, 0.1% SDS, each during 20 min at 60 °C. Membranes were then subjected to phosphoimager analysis using a Typhoon Trio Imaging System  for 2 h. For the P32 labelled RNA probes, first, the DNA fragment for each probe  were cloned in the TOPO-TA Cloning plasmid vector (Invitrogen). Then, according to the sens of the cloning, the 3’ end of each probe were cut with the appropriated restriction enzyme. Thereafter, the transcription of the probe in presence of α-P32 UTP were synthethized using either SP6 or T7 RNA polymerase. Finally, the probes were purified on Sephadex spin-column. In a second step, Blots were re-probed with U6 as a loading control.Total RNAs from the different genotypes were extracted from the dissected gut with TRIzol reagent following the supplier’s instructions (Invitrogen). Denatured RNA samples were run for 1 h  on precasted (10 × 10 cm) TBE urea 10% acrylamide gel (Biorad). We use, for 69. Dried total RNA (10µg for each reaction) were treated with (or without for control) N-cyclohexyl-N’-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulphonate (CMC) for 30 min at 40 °C, and subjected to alkali hydrolysis in the presence of 50 mM Na2CO3 (pH 10.4) at 50 °C during 2 h with shaking every 10 min. The primers of each probes (18S and 28S-rRNA) were radiolabeled using the T4 Polynucleotides Kinases (T4-PNK) and γ-P32-ATP. The 20 mers primers (see 32 5’-labelled primers (the same as for the pseudouridylation reaction) instead of labelled nucleotides. Then, both reactions were migrated in parallel on an 8% acrylamide gel + 8 M urea, at 2000 volts during 2 h. After drying, the gels were then subjected to phosphoimager analysis using a Typhoon Trio Imaging System  for 2 h.Total RNAs from the different genotypes were extracted from the dissected gut with the SV Total RNA Isolation System Kit , followed by a DNase-1 treatment (30 min at 37 °C) to completely remove any DNA trace. Then the total RNA were purified with the NucleoSpin RNA Clean-up kit , and the concentration determined. For pseudouridylation experiments, we follow the protocol of Huang et al. (2016)2 in the posterior midgut. The displayed data represent the means of these two distinct areas. Second, the number of each other specific cell types were counted on the whole gut. We used appropriated genetic markers: for the ISCs, we use Dl-Gal4 driving the UAS-GFP-nls . For the dividing cells, we use two independent approaches (EdU and anti-PH3). For the EdU labelling, the guts were dissected in the PBS and incubated for 1 h in EdU solution (20 µM) at 25 °C 53, using the Click-iT Reaction Imaging Kit . The gut were then mounted in Mowiol and examined to the microscope. For the anti-phospo Histone-H3 (anti-PH3) immunostaining, the guts were dissected in the PBS and fixed for 1 h in 4% PFA. Then washed 3 times in PBST , followed by 4 h incubation in PBST, and then with the primary antibody  at 1/2000  overnight at 4 °C. The day after, the gut were washed 3 times, followed by one hour incubation with the secondary antibody (goat anti-rabbit-Alexa488) at 1/400  at RT, then re-washed again 3 time with PBST and then mounted in Mowiol.First, to determine the number of cells of the different cell types, the gut were dissected in PBS and then fixed in 4% PFA for one hour, followed by 3 washes with PBS and then stained with DAPI for 5 min, and finally mounted in Mowiol. To determine the number of ECs, we count the total number of nucleus stained with DAPI on microscope images taken on two distinct areas of a 33,000 µm56.To quantify the endoreplication of the enterocytes, we used the same fixation protocol as to count the number of enterocytes, except that we precociously and strictly quantify the DAPI-fluorescence according to Weiss et al., 1998Following amplification, flies were harvested on one day. Female flies were maintained with males in fresh food vials for 4 days. On day 4, females were separated from males, and distributed in a cage holding between 200 and 250 females. The cage (50 mm diameter × 100 mm high) is made of plexiglass. The top is covered by a mesh, while the bottom (floor) is closed by a petri dish containing the food medium. The petri dish was replaced every 2 days, and the number of dead flies counted. Note: The lifespan determination of each group of flies and their respective controls (genotypes) have been performed simultaneously, strictly in parallel. Moreover, all longevity experiments have been repeated 2 or 3 times independently.For starvation test, female flies were maintained with males in fresh food vials for 4 days, before testing. On day 4, females were separated from males, and distributed in vials containing 20 females each. Each vial contained a filter, and cotton moisturized with 200 µl of water, to keep the filter moist during the test. The flies were kept at 24 °C in a humid chamber, to avoid dehydration. Dead flies were counted every 12 h.For desiccation test, flies were reared at 25 °C, as usual. Then, female flies were maintained with males in fresh food vials for 4 days, at 25 °C, before testing. On day 4, females were separated from males, and distributed in empty glass vials containing 20 females each, without any filter paper. The vials were transferred and tested at 37 °C, for young flies. However, since 40-day-old flies die rapidly at 37 °C precluding a clear differentiation of the various genotypes, the stress test assay was performed at 30 °C, a milder stress condition. Dead flies were counted every hour.D. melanogaster genome  with TopHat2. Mapped reads were assigned to features with featureCounts 1.5.0-p2 and differential analysis has been done with DESeq2. Three independent biological replicates have been done for each genotype. For the Wild-Type CS and deletion (F4), the RNA-Seq has been performed in the Platform of the I2BC, CNRS, Gif-sur-Yvette, followed by a Bioinformatic analysis performed by Novogene Co . For the overexpression of jouvence (Mex-GS>UAS-jou8M) flies induced by feeding with RU486 (during 7 days) have been compared to non-induced flies (without RU486 feeding). For this last, the complete RNA-Seq analysis  has been performed by Novogene.For RNA extraction, each single hand-dissected gut was immediately soaked in liquid nitrogen and keep at −80 °C. 30 guts were dissected for each genotype. RNA extraction was performed with SV Total RNA Isolation kit  following standard procedures provided by the manufacturer. Thereafter, the total RNA was treated with RQ1 RNase free DNase (Promega) to remove any DNA contamination, and verified by PCR. Then, total RNA were checked for integrity on a bioanalyzer (Agilent). PolyA RNA-Seq libraries were constructed using the Truseq mRNA stranded kit (Illumina) according to the manufacturer recommendations. Libraries were sequenced (Single Read 75 pb) on an Illumina NextSeq500 instrument, using a NextSeq 500 High Output 75 cycles kit. Demultiplexing has been done (bcl2fastq2 V2.15.0) and adapters removed (Cutadapt1.9.1). Reads have been mapped on the https://sbi.postech.ac.kr/oasis2/)70. For the qPCR, and the quantification of number of different intestinal cells, data were analysed statistically using analysis of variance (one-way ANOVA) followed by a TUKEY test, with StatisticaTM software.For the longevity and stress resistance survival curves, log-ranks test was performed using the freely available OASIS software (We declare to have complied with all the relevant ethical regulations for animal testing and research.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Reporting Summary


Transcatheter tricuspid valve repair (TTVR) is a promising technique for the treatment of tricuspid regurgitation (TR). Data comparing the performance of novel edge-to-edge devices (PASCAL and MitraClip-XTR) are scarce.We identified 80 consecutive patients who underwent TTVR using either the PASCAL or MitraClip-XTR system to treat symptomatic TR from July 2018 to June 2020. To adjust for baseline imbalances, we performed a propensity score (PS) 1:1 matching. The primary endpoint was a reduction in TR severity by at least one grade at 30 days.n = 44) was at high-surgical risk (EuroSCORE II: 7.5% [interquartile range (IQR) 4.8–12.1%]) with a mean TR grade of 4.3 ± 0.8 and median coaptation gap of 6.2 mm [IQR 3.2–9.1 mm]. The primary endpoint was similarly observed in both groups (PASCAL: 91% vs. MitraClip-XTR: 96%). Multiple device implantation was the most common form , and the occurrence of SLDA was comparable between the PASCAL and MitraClip-XTR system . No periprocedural death or conversions to surgery occurred, and 30-day mortality  and 3-month mortality  were similar between both groups. During follow-up, functional NYHA class, 6-min walking distance, and health status improved in both groups.The PS-matched cohort (Both TTVR devices, PASCAL and MitraClip-XTR, appeared feasible and comparable for an effective TR reduction. Randomized head-to-head comparisons will help to further define the appropriate scope of application of each system.The online version contains supplementary material available at 10.1007/s00392-020-01784-w. Tricuspid regurgitation (TR) is no longer called “a forgotten valvular disease.” Instead, TR has turned out as a prognostic devastator. Previous cohort studies demonstrated a clear association between significant TR and excess mortality and reduced quality of life (QOL) , 2. Due In this context, we aimed (1) to compare the PASCAL and MitralClip-XTR system concerning the efficacy of TR reduction, and (2) to assess clinical outcomes after the procedures.The study was designed as a retrospective analysis of data from the Bonn registry, which is a single-center, prospective, consecutive database of patients treated at the University of Bonn Heart Center. We identified consecutive patients who underwent a TTVR using the PASCAL or MitraClip-XTR systems from April 2018 to June 2020. The MitraClip-XTR system was utilized as an off-label use. The PASCAL system was available from February 2019 (off-label use) and commercially available since May 2020. All patients had symptomatic TR and were considered as inoperable or at high-surgical risk. After a standardized diagnostic workup including transesophageal echocardiography (TEE), the decision to perform the intervention was taken by the interdisciplinary heart team of the Heart Center Bonn. We excluded patients who underwent a combination of edge-to-edge and annuloplasty technique. This study was approved by the ethics committee of the individual center and was conducted in accordance with the Declaration of Helsinki. All patients participated in the study after written informed consent was obtained.Procedures were performed under general anesthesia with 2D- and 3D-TEE and fluoroscopic guidance. Both the PASCAL and MitraClip-XTR system have been well described previously . After dWe assessed echocardiographic parameters performed at baseline, 30 days, and follow-up . TEE wasThe primary endpoint was a reduction in TR severity by at least one grade at 30 days. Secondary endpoints were implant success during the procedure, 30-day mortality, and 3-month mortality. Implant success was defined as successful delivery and deployment of one or more clips to achieve leaflet approximation and retrieval of the delivery system . ClinicaU test. A paired t test was used to compare a series of variables with regard to symptomatic functional capacity .Categorized variables are presented as numbers and percentages. Normally distributed variables are presented as mean ± standard deviation and compared using t tests. In contrast, non-normal distributed variables are reported as medians and interquartile ranges (IQRs) and compared using the Mann–Whitney Propensity score (PS) was calculated for each patient using multivariate logistic regression that estimates the propensity toward belonging to a specific treatment group (PASCAL versus MitraClip-XTR). This was performed using multivariable logistic regression. Following covariates were included in this PS model: right atrial area, tricuspid annulus diameter, coaptation gap, effective regurgitant orifice area (EROA), vena contracta, TR jet location, and TR grade , 9. We cIn the sensitivity analysis, we used stabilized inverse probability weighting (stabilized IPW) to examine the association of PASCAL with outcomes in this observational study . Weightip values < 0.05 were regarded as statistically significant. All statistical analyses were performed using EZR version 1.37  or R version 3.5.2 .Two-tailed n = 22) or MitraClip-XTR (n = 58) during the study period and were included in the analysis . Baseline characteristics before matching are shown in Supplemental Table 1. Overall, the study patients were mean 78 years old and predominantly female (58%), highly symptomatic  and had a significant burden of comorbidities , which translated into high-surgical risk  and a greater TR  with a larger EROA  compared to those treated with the MitraClip-XTR system. Furthermore, the PASCAL group showed more often TR in the antero-posterior commissure compared to the MitraClip-XTR group .Patients treated with the PASCAL system had a larger right atrial area . Devices were implanted mainly in the antero-septal commissure in both groups, PASCAL and MitraClip-XTR , followed by postero-septal position. Multiple device implantation (≥ 2) was the most common strategy for both PASCAL and MitraClip-XTR systems . On average, the number of devices was significantly lower in the PASAL group . The independent clasping was applied in 19 (86%) patients in the PASCAL group. The occurrence of SLDA was similar between the groups . No surgical conversion or periprocedural mortality occurred.Procedural findings in the overall cohort are summarized in Supplemental Table 2, and those in the PS-matched cohort are shown in Table p = 0.87) or successful implantation .In the sensitivity analysis , similar to the primary findings using PS matching, there were no significant differences between the PASCAL and MitraClip-XTR groups in the primary outcome  or 3-month mortality between both groups . Similarly, in the sensitivity analysis, there were no significant differences between the groups in the 30-day mortality  or 3-month mortality .The 3-month follow-up was completed in 68 (85%) patients in the overall cohort and 36 82%) patients in the PS-matched cohort. Survival curves are depicted for the overall cohort in Supplemental Fig. 2 and for the PS-matched cohort in Fig. % patientp = 0.03). Similarly, there was a significant improvement of health status for the physical component  and the mental component  from baseline to follow-up. With a limited sample size, trends toward an improvement of 6-min walking distance and health status were observed in both groups.With regard to the symptomatic status, functional NYHA class showed a significant improvement in both groups, with the percentage of NYHA class I/II increasing from 5% at baseline to 93% at follow-up in the PASCAL group and 13–82% in the MitraClip-XTR group . A potential explanation for different TR reduction could be SLDA. However, SLDA did not turn out to be pronounced in both groups with each 9%, whereas the earlier studies reported the rates of nearly 7% [Effective and durable reduction of TR is another crucial therapeutic objective of TTVR. We observed a TR reduction by ≥ 1 grade in 93% of patients in the PS-matched cohort, whereas 86% were reported for the Triluminate trial. However, other groups investigating the performance of XTR  or PASCAearly 7% . The preClinical outcome and NYHA functional scale after both types of edge-to-edge repair were promising and comparable to previously published data of both devices that reported 69–85% of patients in NYHA class I/II after 30 days , 22. CouSeveral limitations should be acknowledged. First, the present study was conducted retrospectively, based on a single-center and relatively small sample size cohort. Therefore, a certain patient selection bias might have impacted our results, and the analysis might be underpowered. Nevertheless, we used PS-matching analysis to overcome selection bias and any anatomic imbalance. In turn, the rate of successful TR reduction was consistently observed in the PASCAL and MitraClip groups. Assuming that successful TR reduction is a significant predictor of mortality , 17, theIn this PS-matched cohort, we found that edge-to-edge treatment with either the PASCAL or the MitraClip-XTR device was feasible, effective, and safe in patients with severe tricuspid regurgitation and was associated with clinical improvement. Whether TR reduction is associated with improvement in relevant clinical outcomes parameters such as mortality and rehospitalization has to be answered in future studies. Randomized trials investigating the effect for both devices compared to optimal medical treatment on reducing TR are ongoing . Even more substantial would be a randomized head-to-head comparison between these two TTVR devices and optimal medical therapy.Supplementary file2 (DOCX 15 KB)Supplementary file3 (DOCX 17 KB)Supplemental Figure 1. Study population. During the study period, 80 patients were treated with the PASCAL or MitraClip-XTR systems for the treatment of TR. After propensity score matching, 44 patients (22 PASCAL vs. 22 MitraClip-XTR) were included into the present analysisSupplemental Figure 2. Survival analysis for entire cohort. There was no significant difference in 30-day and 3-month mortality between the PASCAL and MitraClip-XTR groupsSupplementary file1 (XLSX 186 KB)Below is the link to the electronic supplementary material.


For the in vivo study, estrogen deficiency was induced by ovariectomy in 6-weeks-old C57BL/6 female mice, and then randomly given 17β-estradiol (E2) replacements at a low dose (0.01 mg/60 days) and high dose (0.18 mg/60 days), or vehicle treatment. All mice were killed 2 months after treatments, and heart tissues were collected for ex vivo expansion of CSCs. Compared to age-matched healthy controls, estrogen deficiency slightly decreased the yield of CSCs with significantly lower telomerase activity and more DNA damage. Interestingly, E2 replacements at low and high doses significantly increased the yield of CSCs and reversed the quality impairment of CSCs following estrogen deficiency. For the in vitro study, twice-passaged CSCs from the hearts of adult healthy female mice were cultured with the supplement of 0.01, 0.1, and 1 μM E2 in the medium for 3 days. We found that E2 supplement increased c-kit expression, increased proliferative activity, improved telomerase activity, and reduced DNA damage of CSCs in a dose-dependent manner. Our data suggested the potential role of estrogen in maintaining the quality of CSCs, providing new insight into the cardio-protective effects of estrogen.Compared to the age-matched men, the incidence of cardiovascular diseases is lower in premenopausal but higher in postmenopausal women, suggesting the cardio-protective role of estrogen in females. Although cardiac stem cells (CSCs) express estrogen receptors, yet the effects of estrogen on CSCs remain unclear. In this study, we investigated the potential role of estrogen in maintaining the quality of CSCs by  Estrogen plays an important role in maintaining the homeostasis of various tissues/organs –3. EstroEstrogen exerts its biological effects through receptors, including estrogen receptor alpha, estrogen receptor beta, and G-protein-coupled ER, which are present in cardiac myocytes, arterial smooth muscle cells, and endothelial cells , 9. It hRecently, stem cells have been identified in almost all of the tissues/organs, and are known to exhibit a pivotal role in maintaining the homeostasis of the body . Emergin2-treated CSCs provide better cardio-protective effects [in vitro experiments, we herein investigated the role of estrogen in maintaining the quality of CSCs.CSCs, one of the most promising stem cell sources for myocardial repair have been tested in clinical trials and showed beneficial effects –27. It h effects . Howeverin vivo study, 6-weeks-old female C57BL/6 mice were used. For the in vitro study, 10-12-weeks-old female C57BL/6 mice were used. All experiments were approved by the Institutional Animal Care and Use Committee of Nagasaki University, and the experiments were performed following the institutional and national guidelines.For the 2 pellets  of a low dose , or a high dose  were subcutaneously inserted to the mice soon after ovariectomy [ex vivo expansion of CSCs.The 6-weeks-old female C57BL/6 mice were ovariectomized under general anesthesia. The slow-release Eriectomy . Mice reriectomy , 30. All2 incubator. Twice-passaged CSCs were used for evaluations.CSCs were expanded using a method similar to the previously described . In brie4/well) were cultured in 8-well chamber culture slides  coated with 15 μg/ml fibronectin for 3 days and then fixed with 4% PFA for 10 min. After blocking, the cells were incubated with PE-conjugated rat anti-mouse c-kit antibody (eBioscience). The nuclei were labeled with DAPI. The positively stained cells were counted under a fluorescent microscope with 200-fold magnification in twenty randomly selected fields, and the averages from 3 independent experiments were used for statistical analysis.The expression of c-kit in CSCs was evaluated by immunostaining as previously described . In brie4/well) were cultured on 8-well chamber culture slides for 3 days and then fixed in 4% PFA for 10 min. After blocking, the cells were incubated with primary antibodies against Ki-67 (Dako), telomerase reverse transcriptase (TERT) (Abcam), and 53BP1 (Abcam), and followed by the appropriate fluorescence-conjugated secondary antibodies. The cell nuclei were labeled with DAPI. Positively stained cells were counted as described above.The proliferative activity, telomerase activity, and DNA damage of CSCs were also detected by immunostaining . In briein vitro experiments to confirm the potential effect of estrogen on CSCs. Briefly, CSCs were expanded from the atrial tissues of 12-week-old healthy female mice as described above. The same twice-passaged CSCs were cultured in 8-well chamber culture slides with the supplement of 0, 0.01, 0.1, 1.0 μM E2 (Sigma-Aldrich) in the medium, as previously described [We further performed escribed , 28. AftAll of the results are presented as the mean±SD. The statistical analysis was performed by using one-way analysis of variance (ANOVA), followed by Tukey’s test as a post comparison between groups (GraphPad Prism). Differences were considered significant when P<0.05.2 , high dose E2 , or vehicle treatment (Ovx group), respectively. Sham-operated mice were used as healthy controls (Control group). After 2 months of treatments, mice were sacrificed and heart tissues were used for ex vivo expansion of CSCs. Compared with the healthy controls, the uterine weight was significantly decreased in ovariectomized mice  than the Control group (2.78±0.13 x105 cells), there was no significant difference between groups , the expression of c-kit in CSCs was increased by the supplement with 0.01 μM E2 , 0.1 μM E2 , the expression of Ki-67 in CSCs was increased by the supplement with 0.01μM E2 , 0.1 μM E2  and 53BP1, respectively. Compared to the Control group, the expression of TERT in CSCs was significantly decreased in the Ovx group , the TERT-positive cells in CSCs was increased by the supplement with 0.01μM E2  and a high dose (0.18 mg/60 days) to mice following ovariectomy. Estrogen deficiency in the ovariectomized mice was confirmed by the presence of uterine atrophy, which was completely recovered by the replacement with high dose E2. Although CSCs could be expanded easily from the atrial tissues of all mice received different treatments, estrogen deficiency slightly decreased the yield of CSCs with lower telomerase activity and higher DNA damage. All the estrogen deficiency-induced changes in CSCs could be effectively recovered by the replacement with E2 at either low or high doses. The benefits of E2 supplement to CSCs were also clearly indicated by various in vitro evaluations.As the beneficial effects of estrogen to the heart may largely depend on the dosage of E2 , 39, we 2 treatment [2 in CSCs.Previous study by Tang et al. has demonstrated the potent regenerative effects of the CSC-derived c-kit-positive cells through paracrine mechanisms . It has reatment . ERα hasreatment . Agreed Telomeres are specific DNA-protein components present at the chromosomal terminus of mammalian cells. Telomeres elongation is necessary to prevent DNA damage and senescence for allowing the proliferation of the cells. TERT is responsible for the telomeric replication and maintaining their elongations –46. Estr2 levels in mice. Although the bioassay method (e.g. uterine weight) may help to monitor the long-term effects of estrogen replacement using the estradiol-sustained-release E2 pellets, it is asked to identify the optimal dose of E2 supplementation to keep the estrogen at a physiological level for mice received ovariectomy. Second, the adverse effects of high-dose estrogen on other organs needed to be evaluated. Third, we did not measure the cardiac hemodynamics or the cardiac function using a damaged heart as an experimental model, and the telomerase activity was only identified by immunostaining on the expression of TERT. Otherwise, we did not try to further identify the underlying molecular mechanisms on how estrogen maintaining the quality of CSCs.There are several limitations to this study. First, we only used female mice for experiments and did not measure the EIn summary, our data indicated the potential role of estrogen in maintaining the quality of CSCs, which may provide a novel insight into the cardio-protective effects of estrogen. Uncovering the relevant molecular mechanisms may help to develop specific pharmacological compounds that selectively target the estrogen receptors for the treatment/prevention of postmenopausal cardiovascular diseases.S1 Fig2: ovariectomized mice supplemented with low dose of 17β-estradiol (0.01 mg/60 days), HE2: ovariectomized mice supplemented with high dose of 17β-estradiol (0.18 mg/60 days). n = 3 for each group. All data are mean ± SD from 3 independent experiments. *P<0.05. The statistical significance was determined by one-way analysis of variance (ANOVA), followed by Tukey’s test as a post comparison between groups (GraphPad Prism).Uterine weight was measured 2 months after treatments. Control: sham-operated mice, Ovx: ovariectomized mice, LE(JPG)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Participants were randomized to daily DHA plus EPA (2 g/d) or placebo (wheat germ oil) from 10–16 weeks gestation to delivery. Neonatal body composition, fetal growth and length of gestation were assessed. For the 48 dyads with outcome data, median (IQR) maternal BMI was 30.2  kg/m2. In sex-adjusted analyses, n-3 supplementation was associated with higher neonatal fat-free mass  but not with % body fat or fat mass. Birthweight for gestational age z-score  was higher, and gestation longer  vs. 39  weeks, p = 0.02), in the treatment vs. placebo group. Supplementation with n-3 PUFA in women with OWOB led to higher lean mass accrual at birth as well as improved fetal growth and longer gestation. Larger well-powered trials of n-3 PUFA supplementation specifically in pregnant women with OWOB should be conducted to confirm these findings and explore the long-term impact on offspring obesity and cardiometabolic health. Maternal obesity, a state of chronic low-grade metabolic inflammation, is a growing health burden associated with offspring adiposity, abnormal fetal growth and prematurity, which are all linked to adverse offspring cardiometabolic health. Higher intake of anti-inflammatory omega-3 (n-3) polyunsaturated fatty acids (PUFA) in pregnancy has been associated with lower adiposity, higher birthweight and longer gestation. However, the effects of n-3 supplementation specifically in pregnant women with overweight and obesity (OWOB) have not been explored. We conducted a pilot double-blind randomized controlled trial of 72 pregnant women with first trimester body mass index (BMI) ≥ 25 kg/m The prevalence of pre-pregnancy overweight and obesity (OWOB) is increasing worldwide  and is cMaternal obesity-associated inflammation has been found to be associated with altered placental lipid metabolism and insulin signaling that are thought to lead to excessive nutrient transport, resulting in increased fetal fat accretion ,11. On tPolyunsaturated fatty acids (PUFA) can modulate inflammation, insulin sensitivity and lipid metabolism, and the PUFA profile during pregnancy has been associated with length of gestation and offspring adiposity . A recenAnimal studies ,12,17,182 to investigate whether n-3 supplementation starting in early gestation (<16 weeks) would improve maternal insulin sensitivity and reduce inflammation, the co-primary outcomes of this trial. We have previously reported on these outcomes and shown that n-3 PUFA supplementation reduced systemic and placental inflammation, though there was no significant effect on insulin resistance [We conducted a pilot double-blind RCT in healthy women with body mass index (BMI) > 25 kg/msistance . We havesistance . The objective of the present study was to explore the preliminary efficacy of n-3 PUFA supplementation on neonatal adiposity (primary outcome), and fetal growth and length of gestation (secondary outcomes). Neonatal adiposity was a secondary outcome measure in this pilot RCT, though this measure will be evaluated as the primary outcome for this manuscript. We aimed to explore preliminary evidence of efficacy and help inform future larger well-powered trials of n-3 supplementation in pregnant women with overweight and obesity. Fetal growth and length were not specified as outcomes in the original study design and thus are secondary analyses of the data. Therefore, the present study is not powered to assess the effects of n-3 PUFA supplementation on these outcomes. Given their anti-inflammatory and insulin-sensitizing properties, we hypothesized that n-3 PUFA supplementation in women with OWOB would result in lower adiposity, higher fetal growth, and longer gestation. This study was supported by an American Recovery and Reinvestment Act (ARRA) 2-year grant from the National Institutes of Health RHD057236.2 at MetroHealth Medical Center/Case Western Reserve University. Participants were randomized in early gestation (<16 weeks) to either oral n-3 supplements containing 800 mg docosahexaenoic acid (DHA) and 1200 mg eicosapentaenoic acid (EPA) for a total of 2000 mg of n-3 PUFA, divided into four capsules, or matching placebo capsules contained wheat germ oil. All subjects were instructed to take two capsules twice a day from enrollment until delivery. Details of the study design, inclusion and exclusion criteria and methods of the trial were described previously [2 at the first antenatal visit without evidence of existing metabolic disorder such as hypertension, diabetes or hyperthyroidism. Of the 620 women who were screened, 156 agreed to be enrolled in the one-week run-in, and of these women, 84 failed the run-in as they did not return or had taken less than 50% of the placebo capsules. The 72 participants who passed the compliance run-in were randomized using a computer-generated randomization table . Women were allocated to study arms by Eminent according to the randomization list and we received identical packets with unique study numbers for each participant. Eminent Corporation had no contact with participants and had no role in either study recruitment or its execution. Of the 72 women who passed the run-in, 23 were lost to follow-up due to participant burden in light of the various metabolic studies which were part of the protocol. Forty-nine women completed the trial. For the current analysis, we additionally excluded one mother-child dyad due to fetal demise. We included n = 48 mother-infant dyads in this analysis  to the nearest 0.01 kg. BMI was calculated as kg/mAt the first and second visits, participants completed a semiquantitative food-frequency questionnaire (FFQ) which was modified for use in pregnancy from a well-validated instrument used in several large cohorts of non-pregnant adults , and valv/v) chloroform:methanol and washed with 0.88% KCl per the method described by Foch et al. [Maternal blood was collected at V1 and V2. Trained research staff at the CRU immediately separated the plasma from erythrocytes and stored aliquots in liquid nitrogen. Details of the fatty acids (FA) analysis were described previously . Brieflyh et al.  Fatty acTM Body Composition System . The PeaPod was administered twice for each infant. If the percent of body fat differed by >0.2%, the test was repeated a third time. For each outcome, the average of the two closest measures was used. Using pressure-volume equations, FM and FFM were estimated to provide BF% which was derived as 100 × FM/(FM + FFM).We assessed body composition two ways: (1) we estimated neonatal FM from BW, length, and flank skin fold thickness (SFT) using a previously validated anthropometric equation . Fat frez score (BWGA-z) and birth length for gestational age z-score (BLGA-z). All z scores were calculated using the Fenton 2013 clinical calculator [Neonatal anthropometric measurements, including BW, length, and flank SFT, were obtained within 72 h of delivery. Detailed methods for neonatal anthropometric measurements have been described previously . Fetal glculator . Also usLength of gestation was calculated based on last menstrual period and confirmed based on dating ultrasound.p-value < 0.05 was considered statistically significant for all analyses. Data were analyzed using intention-to-treat. Descriptive statistics were used to characterize maternal and neonatal characteristics by treatment group after testing for normal distribution. We used the Wilcoxon-rank sum test to compare dietary intake and FA concentrations between treatment groups. We used the Wilcoxon signed-rank tests to determine if dietary intake and FA concentrations following supplementation were significantly different from baseline within each treatment group. We assessed Pearson or Spearman’s correlations between Peapod and anthropometric measures of body composition. We used the Student t test or Wilcoxon-rank sum test for continuous variables, and the χ2 test for categorical variables, for comparisons of outcome measures between treatment groups. We then performed multivariable linear regression analyses to estimate differences between treatment groups (placebo as reference) in measures of body composition adjusted for sex since neonatal body composition differ in males vs. females. We did not adjust for maternal characteristics since this was a RCT and baseline characteristics were similar between groups. Finally, we use linear regression analyses in stratified analyses to determine whether estimates of the difference between treatment groups (placebo as reference) in the outcome measures differ by first trimester BMI category (overweight vs. obese), maternal n-6/n-3 baseline dietary intake (below vs. above median intake), and by offspring sex. Prior studies have shown that n-3 supplementation may be beneficial specifically in women with overall lower dietary quality (low dietary and plasma n-3 PUFA) and with the highest metabolic dysfunction. Furthermore, animal data have shown sexual dimorphism in the effects of maternal dysmetabolism, inflammation and overnutrition on fetal development with males being more susceptible to the in-utero environment. Therefore, we hypothesized that male offspring as well as women with obesity and with the highest dietary n-6/n-3 intake, a marker of dietary inflammation, would benefit most from n-3 supplementation.All statistical analyses were performed using Stata/SE version 15.1 . A two-sided p = 0.03) and more likely to be White . The baseline characteristics of the participants are summarized in 2 vs. 33  kg/m2; p = 0.04) and were more likely to be nulliparous . Among the 48 included participants, the mean ± SD maternal age at enrollment was 26.9 ± 5 years. The median and interquartile range (IQR) maternal BMI and gestational age (GA) at recruitment were 30.2  kg/m2 and 14.8  weeks. Amongst all the women, 44% were white, 35% were African American, 50% were overweight and 50% had obesity. There were no differences in maternal characteristics between placebo and treatment groups. Five participants were diagnosed with gestational diabetes melitus (GDM) and were diet-controlled. Among the children, 52% were female and 48% were male.Compared to the women who were lost to follow-up, the women who completed the study had lower baseline BMI (median (IQR): 30  kg/mp > 0.05).In p < 0.05). The ratios of total n-6/n-3 and of AA/DHA+EPA were lower in the n-3 vs. placebo group at V2 .Plasma FA concentrations at V1 and V2 by treatment group are presented in p < 0.001), FFM , and BF%  measures from the validated anthropometric method and PeaPod method were highly correlated. Results using the anthropometric method were available for all 48 newborns and presented in FM   in adjusp = 0.24, p = 0.02). There were no LGA children in either group. The percentage of SGA infants was 8% in the n-3 group and 17% in the placebo group, though the difference was not significant (p = 0.38).Newborns of mothers supplemented with n-3 PUFA during pregnancy had higher BW  and BWGA-z (n-3: −0.17 ± 0.67 SD units vs. placebo: −0.61 ± 0.61 SD units) compared to control. They also had higher birth length though BLGA-z was similar between the two groups . There were no children born post-dates in this study.Length of gestation was one week longer among women in the n-3  weeks) vs. placebo  weeks) group  and FFM  . There wNewborns of women with a high (above median) early pregnancy n-6/n-3 dietary intake supplemented with n-3 vs. placebo had higher BW , BWGA-z  and FFM  . There wSupplementation with n-3 PUFA had a significantly greater effect on male compared to female neonates. The effect of n-3 supplementation during pregnancy on BW  and the anthropometry-derived neonatal FFM  was stronger in male compared to female neonates . The effIn this randomized pilot study of a high-risk cohort of women with OWOB, we report for the first-time preliminary evidence of efficacy potential of n-3 supplementation. We showed that daily supplementation with 2 g of DHA and EPA starting early in pregnancy (~14 weeks) until delivery resulted in a 7-day longer length of gestation, 343 g higher birthweight, and 0.44 SD unit higher BWGA-z score. Interestingly, the higher fetal growth was not the result of higher adiposity, but instead reflected a higher fat-free mass accrual.Previous studies have suggested that infant adiposity at birth rather than BW is more sensitive to maternal dysmetabolism; therefore, evaluating body composition might provide better insight into maternal-fetal programming mechanisms. Here, we showed that offspring of women with OWOB supplemented with n-3 PUFA have higher FFM at birth, where FFM represents the combination of muscle mass, water and bone mineral content (BMC). Our findings are consistent with prior RCTs of n-3 supplementation in pregnant women which showed that higher maternal DHA status was related to higher offspring FFM at birth and early childhood without a concomitant change in FM or BF% ,27,44. HVinding  et al. sThe mechanisms via which n-3 PUFA might influence FFM accrual are unclear. Animal studies suggest that n-3 PUFA can promote calcium absorption and osteoblast formation which could increase bone density, growth and elongation ,47. Addiin-utero milieu even earlier in pregnancy. Several observational studies have shown associations between higher n-3 status in pregnancy or lactation and lower offspring adiposity in childhood ,16,50. Win-utero metabolic milieu of OWOB pregnancies as we have previously demonstrated in this cohort [in-utero effects on offspring body composition [We found that BW was higher in the n-3 PUFA group, and that the higher BW was not attributable only to longer gestation as has been previously suggested . These fs cohort ,36. Supps cohort  et al. ss cohort ; howeverposition . It is nMakrides  et al. dOur findings are in line with a Cochrane systematic review  that shoResults of our stratified analyses showed that the effects of n-3 supplementation were only evident in women with obesity or with a high dietary n-6/n-3 ratio. These findings are consistent with prior RCTs which showed that longer gestation and reduced risk for preterm birth as a result of n-3 supplementation were most evident among women with low n-3 PUFA concentration and with low dietary n-3 intake ,29. Pre-in-utero conditions may be amplified in the male fetus [We also found significant interactions between n-3 supplementation and offspring sex whereby n-3 supplementation had stronger effects in males vs. females. This is contrary to previous studies that have not found significant sex-dependent effects in the setting of n-3 supplementation ,27,44. Ale fetus . Rodent le fetus . Althougle fetus .Our findings must be interpreted within the context of study limitations. As a pilot study, it was not intended to be adequately powered to show significant differences between groups in our outcomes of interest. However, the current pilot study’s sample size was optimal in order to inform a future large RCT with 80% power to detect small-to-moderate effect size in fetal adiposity . Althougin-utero milieu early in pregnancy in metabolic programming. We used validated measures of body composition and evaluated the effect of n-3 supplementation specifically on FFM and FM, which have different effects on health. Finally, our cohort was racially diverse increasing the generalizability of our findings to more vulnerable populations.Despite these limitations, our study has several strengths. This is the first pilot study of n-3 supplementation specifically in a high-risk cohort of pregnant women with OWOB investigating newborn body composition, fetal growth and length of gestation. The limitation of supplementation studies is that usually the dietary fat content is not controlled which can lead to confounding. Here we assessed dietary intake in both treatment groups and showed that both groups’ diets were similar in the year leading to pregnancy as well during pregnancy. Therefore, the observed differences were likely not due to differences in dietary n-3 PUFA intake or to changes in their diet throughout pregnancy. We evaluated the response to n-3 supplementation by measuring maternal n-3 and n-6 plasma concentrations during the study. The concentrations of DHA+EPA was 36% higher in treatment vs. placebo at V2 which suggest good compliance to treatment assignment. We also started supplementation earlier (~14 weeks) than previous studies which is a strength given the role of the Pre-pregnancy obesity is the strongest independent risk factor for child obesity and complications related to increased risk for preterm births. Women with pre-pregnancy obesity also have a low n-3 relative to n-6 PUFA status, which has been associated with preterm birth. This pilot study provides preliminary evidence that supplementation with n-3 PUFA during pregnancy in women with OWOB for ~25 weeks may increase fetal fat-free mass accrual, improve fetal growth, and increase length of gestation. Improving the n-3 status in pregnancy may have value as a prophylactic intervention in some women, particularly those with high-risk pregnancies such as women with obesity. Larger adequately powered trials of n-3 supplementation or dietary intervention specifically in women with OWOB starting before conception or early in pregnancy should be conducted to confirm these findings and explore the long-term impact on offspring adiposity and cardiometabolic health.